PB83-149328
Mutagenic and Carcinogenic Potency of
Extracts of Diesel and Related
Environmental Emissions
Summary and Discussion of the Results
(U.S.) Health Effects Research Lab.
Research Triangle Park, NC
IS 81.
US. fapvfn&ri of Commerce
Teckrecai Information Service
-------
TECHNICAL REPORT DATA
(flene read Insirucr:em on the reverse before complti'
1. REPORT NO.
FPA-600/J-81-605
JOURNAL ARTICLE
PB83-1U9328
4. TITtE AND SUBTITLE
JOURNAL ARTICLE: Mutagenic and Carcinogenic Potency of
Extracts of Diesel and Related Environmental Emissions:
Summary and Discussion of the Results
5. REPORT DATE
1981
6. PERFORMING ORGANIZATION CODE
7. AUTHORIS)
8. PERFORMING ORGANIZATION REPORT NO.
Stephen ftosnow and Joel "ten Lewtas
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Genetic Toxicology Division
Health Effects Research Laboratory
US Environmental Protection Agency
Research Triangle Park, NC 27711
to. PROGRAM ELEMENT NO.
ACSL1A
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Research and Development
Health Effects Research Laboratory
US Environmental Protection Agency
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-600/11
18. SUPPLEMENTARY NOTES
Environment International 5: 425-429, 1981
16. ABSTRACT
The proposed conversion from gasoline powered automobiles to diesel powered
vehicles has prompted the Environmental Protection Agency to evaluate the potential
health effects associated with exposure to diesel emissions. At present, there is no
direct epldemiological link between this exposure and human'health. Therefore, a
research program was constructed to compare the health effects associated with diesel
emissions with those from other emission sources for which eoidemiological information
was available. The emission sources chosen were cigarette smoke, roofing tar, and
coke oven. An additional comparative emission source which was evaluated was a
gasoline catalyst engine. Respirable particles from a variety of combustion sources
have the potential of being carcinopenic and mutaqenic. The objective of these
studies was to determine the relative biological activity of the organic material
adsorbed on these particles in both In Vitro mutagenesis and In Vitro and In Vivo
careinogenesis bioassays.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Fkld/Gioup
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS < Thil Report I
Unclassified
21. NO. OF PAGES
JO. SECURITY CLASS (Tillpigtl
Unclassified
22. PRICE
EPA F«rat 2220-!l (R««. 4-77) PMCVIOUS RDITION II O«»OUKTC
-------
Emironmcnl Inferrmnvtiul, Vol. 5. pp. 425-429. 1981
Printed in ibc USA.
0160-4120/RI /049425-05S02.00/0
1982 Pergamon Press Ltd.
EPA-600/J-81-605
JOURNAL ARTICLE
MUTAGENIC AND CARCINOGENIC POTENCY
OF EXTRACTS OF DIESEL AND RELATED
ENVIRONMENTAL EMISSIONS:
SUMMARY AND DISCUSSION OF THE RESULTS
Stephen Mesnow and Joellen Leyvtas . .'....".
Ht.'lth Effects Research Laboratory. US Environmental Protectioo Agency. Research Triangle Park.
Noun Carolna 27711. USA . .
UK proposed conversion from gasoline powered automobiles todicsel powered vehicles has prompted the
Environmental Protection Agency to evaluate the potential health effects asstviatcd with exposure to.
diesel emissions, At present, there is no direct cpidcnliological link between this exposure an.) human
health. Therefore, a research program was constructed to compare the health effects associated with dicscl
emissions with those from other emivsion sources for which cpidcmiological information was available.
The emission sources chosen were cigarette smoke, roofing tar. and coke oven. An additional comparative
emission vturcc which was evaluated was a gasoline catalyst engine. Rcspirabb particles (rom a variety of
combustion >ourccs have the potential of being carcinogenic and mntagcnic. The objective of these studies
was to determine the relative biological activity of the organic material adsorbed »n these particles in both
1/1 mm mui.igcncsis and in ii/ro and m riro carcmogcnesis hioaxsays. The organic extracts from the
following series of emission sources were quantitatively hi«a.ss£>cd in a matrix of tests for their
carcinogenic and mutagcnic activity: (1) a light-duly Oldsmobilc dicscl 3M) engine: hinturiufn; miloiic recombination
in .Vu<-e Ivmphoma cells. Balb/c 3T3 mouse embryo
fibroblasls aind C'HO cells: viral enhancement of SHI: cells: oncogcnic transformation in .Balb/c 3T3
cells: and skin tumor initiation in SLNC'AR and CS7 black mice. The results of this lest mains arc
discussed.
Introduction
The propo'sed conversion from gasoline powered aulo-
mobilcs to diescl powered vehicles has prompted ih;
Environmcnial Protection Agency to evaluate the poten-
tial health effects associated with exposure to dicsel
emissions i( Ember. 1979). At present, there is no direct
epidemiologica! link between this exposure and human
health (EPA, 1978). Therefore, a research program was
c::n.strucle<. to compare the health effects associated
with -liescl emissions with the ; from other emission
sou.-sX-!; for which epidemiological information was
avail itric. The emission sources chosen were cigarette
smoke, roofing tar. and coke oven. An additional com-
parative emission source which was evaluated was a
gasc'.ine iitalyst vehicle. - •• . •-
Respi.:aMe particles from a variety of combustion
sources har: the potential of being carcinogenic and
mutagenic (Waters el at., 1979). The objective of these
studies was to determine the relative biological activity
of the organic material adsorbed on these particles in
both in vitro and in vivo mutagenesis and carcinogenesis
bioassays. The organic extracts from the following series
of emission sources were quantitatively bioassayed in a
matrix of tests for their carcinogenic and mutagcnic
activity: (1) a light-duty Oldsmobile dicscl 350 engine,
(2) a heavy-duty Caterpillar 3304 diesel engine. (3) a
light-duty Datsun Nissan 220C engine, (4) a Volksw-a-
gen lurbocharged Rabbit diesel engine. (5) cigarette
smoke, (6) roofing tar, (7) coke oven, and (8) an un-
leaded gasoline catalyst Mustang II. The collection,
characterization, and description of these samples arc
described by Lcwtas el at. (1981).
The test matrix consisted of the following bioassays:
reverse mutation in Salmonella typhimurium; mitotic
recombination in Saccharomyces cerevisiae', DNA
425
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426
breakage in Syrian hamster embryo cells (SHE); sister
chromatid exchange in Chinese hamster ovary (CHO)
cells; gene mutation in L5178Y mouse lymphoma cells.
Balb/c 3T3 mouse embryo fibroblasts. and CHO cells;
viral enhancement in SHE cells; oncogenic transforma-
tion in Balb/c 3T3 cells; and skin turner initiation in
SENCAR and C57 black mice.
The potency of complex mixtures of organic com-
pounds in biological systems will depend on the uptake,
distribution, metabolism, and binding of each compo-
nent of the mixture in each biological system; the specific
biological endpo'it (e.g.. gene mutation): the sensitivity
of each biological system to the individual components
of the mixture; the possible interactions, both synergis-
tic and antagonistic, which arise from the exposure to
multiple agents; and the type of quantitative method
which is applied to the data. Differences in these param-
eters will result in altered potency. •
Aware of these problems and shortcomings, we have
attempted to correlate biological end effects from a
variety of carcinogenesis and mutagencsis bioassays with
the nature or source of a series of combustion samples.
The: purpose of this comparative study is.to evaluate
each of the in vitro bioassay systems in comparison with
each other and with the in vivo system. This paper will
summarize and discuss the results of the bioassays re-
ported in the session on the Mutagenic and Carcino-
genic Potency of Extracts of Diesel and Related En-
vironmental Emissions (Claxton, 1981; Mitchell ei al.,
1981; Casto el al.. 1981; Curren et al.. 1981; Slaga el al..
1981).
Results and Discussion
Quaniitaiion method
General procedures. The method for relating effect with
dose depends on the bioassay being considered. The
bacterial mutation system of Ames (AMES), the sister
chromatid exchange bioassay in Chinese hamster ovary
cells (SCE), the mammalian cell mutagencsis bioassay in
L5178Y mouse lymphoma cells, (L5178Y) and the mouse
skin lumor initiation bioassay (TUMOR INITIAT) gave
good dose-response relationships and the slope of the
linear portion of the dose response curve, was chosen for
potency estimation (Table 1). The Balb/c 3T3 mutagen-
csis and oncogenic transformation -;ioassays (BALB)
did not give good dose response with the samples tested
and closes chosen. A quantitatipn method .was therefore
applied which utilized the lowest effective concentration
te- ted (LF.CT) which exerted a biological response. It is
to be emphasized* however, that with pure agents and
more closely spaced doses the Balb assay does respond
in a dose-related fashion. The viral enhancement bioas-
say in Syrian hamster embryo cells (VIRAL EN-
HANCE) gave dose related response information in
terms of absolute frequency but for comparison to the
Stephen Nonowand Joellen Leu. las
Table I. Comparative potency: Quamitation method.
Mulagene&is
CarcinogcnCMs
VIRAL TUMOR
Bioassoys: AMES SCK L5PSY BALB ENHANCE BALB INITIAT
. Quanli-
lalion
Mcihnl: Slope Slop* Slope LECT LECT LF.CI Slope
LECT= Lowest effective concentration tested.
Slope Aslope of the linear portion of the dose-response curve.
Balb system and due to the kind of statistics applied to
the assay, the LECT method was chosen. It is possible
that the two methods, slope and LECT. when applied to
the same data could give dissimilar relative rankings.
Bioassay results in Saccharomyces cerevisiae. DNA
damage in SHE cells, gene mutation in CHO cells, and
skin tumor initiation in C57 black mice were not utilized
in this analysis due to marginal resu'.'.s.
Nornia/i:ation of Jala. In order to reduce or normalize
the potencies in the bioassays to a common denomina-
tor, the following system was applied: the activity of the
Nissan diesel sample regardless of bioassay type or
quanlilalion method used was given a value of 100. All
the results of other emission samples were then related
to the Nissan sample. For example, in the Ames bioas-
say. Strain TA-98 less metabolic activation, the potency
of Nissan and Oldsmot'Ic by the slope.method was
1225 and 615 revcrtants/ lOO fig. respectively. Assuming
the Nissan potency equals 100 by simple mathematical
relationship, the Oldsmobile sample equals 50 or one-half
the potency of the Nissan sample. In the case of a LECT
potency, the inverse relationship is applied. For example
if the LECT for Nissan is 75 /»g/ml and that for Cat is
300 jtg/ml. assuming Nissan equals 100. then Cat equals
75/300 X 100 or 25, one-quarter the potency of the
Nissan sample. Using this kind of analysis, the absolute
data was converted to normalized values. The reason the
Nissan was chosen as the sample to normalize to was
that it was the only diesel sample tested in all bioassays
which gave positive results.
Comparative rankings
Gene mutation assays. A comparison of the test results of
the eight samples plus the positive control in the three
gene mutation assays is found in Table 2. The three j;ene
mutation assays compared were Ames strain TA-V8.
L5178Y at the TK*'~ locus, and BALB 3T3 at the
ATPase locus. Each system was performed without
(-MA) and with ( + MA) metabolic activation which
consisted of an Aroclor-1254 induced rat hepatic.S-9.
The emission samples were previously described and are
abbreviated as Cat for heavy duty Caterpillar; Nissan;
Olds for Oldsmobile 350; VW Rabbit for the
lurbocharged diesel Rabbit;-Mustang for the unleaded
catalyst gasoline engine; cigarette for standard cigarette
smoke condensate; coke for coke oven emissions; Roof
-------
i Diesel and related environmental emissions
427
• Table 2: Comparative rankings: dene mutation assays.
Table 3. Comparative rankings: DNA damage assay.
AMES'
Sample
Diesel:
Gasoline:
Comparative
Sources:
Standards:
Cat
Nissan
Olds
VWRab
Mustang
Cigarette
Coke
Roof Tar
B( Olds > Rabbit>
Cat. When the gasoline sample is included, the rankings
are: Ames: Nissan > Olds> Rabbit > Mustang> Cat;
mouse lymphoma: Nissan>Olds>Mustang>Rabbit>
Cat. Both of these cell types lack the oxidalive enzymes
required for the activation of mutagens or carcinogens.
The diesel and gasoline samples show primarily direct
acting activity. Balb 3T3 cells possess the en/yincs re-
auired for the activation of carcinogens especially poly-
cyclic aromatics (Curren el al.. 1981) and therefore one
cannot compare all these systems across the board and
expect similar results. Comparison of the thr-x systems
with metabolic activation showed the relative ranking
(with some exceptions) to be: Nissan > Olds> Rabbit >
Cat.
The comparative sources samples generally show more
metabolic activation dependence for maximal effect and
less quantitative correlation in the gene mutation assays.
DNA damage assay. The mobile source samples tested
for sister cliromatid exchange in CHO cells, without
activation. (Table 3) gave the following ranking: Nissan
> Rabbit, Musiang>Ca:, Olds.
In the presence of metabolic activation, the SCE
results with the comparative sources ranked in similar
order with the mouse lymphoma results: roof tar>cokc
> cigarette.
Onnigenic transformation. The results from assays which
relate to the transformation of cells in culture are found
in Table 4. Both of ihese sysierns were evaluated by the
SCE(CHO)'
Sample
Diesel:
Gasoline:
Comparative
Sources:
Standards:
-• •
Cat
Nissan
OUs
VW Rah
Mustang.
Cigarette
Coke
Roof Tar
B Rabbit, Mustang* Cat, Olds, while the
comparative samples rank: roof tar > coke > cigarette.
Mouse skin tumnrigencsix. The results .obtained in the
SENCAR mouse skin tumorigenesis experiments for
tumor initiators have been compared at 14 weeks after
treatment (interim score) for all the samples and at 21
Table 4. Comparative ranking..-.: Oncogenic• transformation assays.
Viral
Sample Enhancement (Sill
. Diesel:
Gasoline:
Comparative
Sources:
Standards:
Cat
Nissan
Olds
VW Rab
Mustang
Cigarette
Coke
Roof Tar
1»(U)P
MNNCi
0
100
0
50
50
•;'200
KOO
1040
50000 '
31150
BALB3T3
E( -MA
1.3
100
1.9
NTh
100
r
10
i
—
HOO
J-MA"
0
100
0
NT
2000
1
I
500
16700
—
'"Metabolic activation by an Artvlor-1254 induced rat hepatic S-*
*Noi tested.
* Testing incomplete at thix time.
-------
42R
Stephen Nc-jnow and Jocllcn Lc»las
Table 5.-. Comparative rankings: SENCAR mouse skin tuniurigcncsis.
Tumor Initiation
Interim Score' Final Score
Sample
Diesel:
(iasolinc:
Comparative
Souices:
Standards:
Cat
Ni.ssan
Olds
VW Rah
Mustang
Cigarette
Coke
Roof Tar
B(o>P
Ranking
0
MX)
45
35
0
71
17900
Potency h 'Potency1"-'
0.00 0.00
0.25X
0.115 0.145
I
(MWO
000
O..W
0.182
46.2 71.6
Table 7. Relative rankings of mobile source .samples.
Bioassav
Rank Order
•"Interim score 1.4, weeks after treatment.
hPapillomx\/mouNe/nig., -•
'Testing incomplete at this lime.
week?, of treatment (final score) for .three samples (Table
5). The interim score is represented here in both abso-
lute ti.-rms (papillomas/mouse/mg) and in the normal-
ized rankings while the final scoring for comparison is
in absolute terms. There are, as expected, differences in
the slope potencies from incomplete vs completed
experiments hut those differences do not exceed 2-fold
in the Olds and K(a)P samples. The Cat and cigarette
samples were negative in this assay system up to 10.000
jig/mouse. The lack of activity of the cigarette smoke
condensate may be due in part to the method of sample
collection. This sample is not an organic extract of
particles as are the other samples and is therefore com-
paratively much less concentrated. This may explain the
very low percentage of benzofujpyrene found in the
cigarette smoke condensate sample (Table 6). The Olds.
Table 6. Correlation between organics anJ tumor initiation.
Mouse Skin
Percent Organic ng B( a IP/ Tumot
lixtractablc mg k\t. Initiation'*
Diesel:
Gasoline:
Comparative
Sources:
Standards:
Cat
Nissan
Olds
VWRab
Mustang
Cigarette
Coke
Roof Tar
U99
-
2
1173
2
26
103
. 478
KH9
10*
0.00
0.25S
0.115
I"
0.09
0.00
0.307
O.IK2
46,2
AMIS'
L5I78V
S0:.' .
Viral Ilnharicemeht'
Tumor Initiation1' •
Nissan > Mustang. Olds. Rabbit » Cat
Nissan > Olds > Rabbit > Mustang
Nissan > Rabbit * Cat. Olds
Nissan > Mustang. Rabbit '*• Cat. Olds
Nissan > Olds > Mustang > Cat
'In the presence of an Aroelor-1254 induced rat hepatic S-^. :
""Mouse skin tumor initiation in SF.N'CAR'inice 14 weeks after treat-
Mustang, and roofing tar activities were less than Nis-
san while the coke oven sample was slightly higher. The
ranking of the mobile source samples was: .Nisw.r»>-
Olds > Mustang * Cat.
A comparison of the amount of B(u)P per milligram
extract, and the potencies of those samples.as. skin tumor.
initiators is found in Table 6. There is little correlation
observed, except for the Olds sample.'.between ng
B(a)IJ/mg extract and papillomas/riiouse/mg extract.
, . « • •- •
Comparison across ten systems • • •
..The relative rankings of the mobile source samples
are listed in Table 7. These results are from those
bioassays performed in the presence of exogenous
metabolic activation.
There is a consistency in these results with the Nissan
sample the most potent and the Cat sample the least
potent in all hioassays. . .
The relative rankings of the comparative source sam-
ples is found in Table R. In three of five systems, the
identical rank order of roof tar>coke>cigarette is
found, and in all five systems, the cigarette sample was
the least potent.
The normalized rankings for 7 bioassays, are com-
pared in Table 9. The results presented are from those
bioassays performed in. the presence of exogenous
metabolic activation including Ames, SCE. L5178Y. and
Balb-mutagcnesis and transformation. The quantitative
results from the mobile source samples show a general
overall consistency! Cut, a very weak sample in Ames is
inactive in SCE, viral enhancement, Balb transforma-
tion, and mouse skin tumor initiation. Olds, weak, in
Ames. L5178Y. Balb mutation, and mouse skin tumor
initiation is inactive in SCE, viral enhancement, and
BALB transformation. In three of the four assays for
Table R. Relative rankings of comparative source samples.
Bioassav
Rank Older
AMI-S*
L5I7KV
SCU"
Viral linhancemenl
Tumor Initiation*1
Coke > Roof Tar. Cigarette
Roof Tar > Coke > Cigarette
Roof Tar > Coke * Cigarette
Root Tar > Coke > Cigarette
Coke > Roof Tar > Cigarette
'Interim '.ore 14 weeks after treatment (papillomas/mouse/mg).
'Testi'.'s incomplete at this lime.
•In the presence of an Aroclor-1254 induced rat hepatic S-9.
. *"Mou.se .skin tumor initiation in SliNCAR mice 14 weeks after treat-
ment. ...
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* Diesel anij related environmental emissions
429
Table 9. Comparative potency rankings.
Mutagenesis
Carcinogenesis
VIRAL TUMOR
AMES' SCE' L-5I78Y' BALB' ENHANCf BALB' INITIAT"
Diesel:
Cat
Nissan
Olds
VWRab
4.3
100
23
22
0
100
0
50
lc
100
64
50
15
100
58
NT"
0
100
0
$0
0
100
0
NT
0
too
45
I
Gasoline:
Comparative
Mustang 25
7500
50
*ln the presence of an Aroclor-1254 induced rat hepatic S-9.
'Mouse skin-tumor initiation in SUNCAR mice after 14 weeks of treatment.
'Testing incomplete at ihis lime.
JNol tested.
2000
35
Sources:
Standards:
Cigarette
Coke
Roof Tar
Wall"
7
IK
7
1112
0
44
291
1750
21
339
850
189
300
15
7500
25.000
200
XOO
1040
50.000
I
1
500
16.700
0
119
71
17.900
which test data is available. SCE; L5I78Y. and viral
enhancement, the Rabbit sample gave almost identical
quantitative results. The Mustang sample gave similar
results in Ames. L5I78Y, and mouse skin tumor initia-
tion, while markedly dissimilar results were obtained in
the Balb assay.
With the comparative source samples, there- is little
agreement between the quantitative results from these
bioassays with the exception of the roofing tar sample in
L5I78Y. viral enhancement and Balb transformation.
In theory, gene mutation and skin tumor initiation
arise from similar one hit. single process, irreversible
mechanisms and should give similar results Assuming
equal toxicity and mutageri/carcinogen transport/
activation by the various cell types. A comparison of the
results of the mobile source samples in Ames and
LSI78Y gene mutation with those results in mouse skin
tumor initiation seem to support this hypothesis.
In conclusion, a series of extracts from diesel and
gasoline emission samples were evaluated in a battery of
bioassays and a broad general agreement was found
among most of the bioassays. Similar experimentation
. with.the cigarette, coke oven, and roofing tar samples.
produced dissimilar results. Additional experimentation
and analysis will continue in this .important area of
environmental mutagencsis and carctnogenesis.
References
Casht. B. O.. Hatch. Ci. G.. Huang. S. I... Lewlas...!. I... Nesnow. S,.
and Waters. M. I). (19XU Mulaiynic and carcinogenic potency of
extracts of diesel anj related environmental emissions: In turn
mulagcncsis and oncogcnic transformation. Kminm. int. 5. 403.
Claxlon. L. (l9S|j Muiagcnic and carcinogenic potency of extracts of
diesel and related environmental emissions: Salmonella typltitriurtum
assay. Knriron. lit: 5. 3X9.. •
Curren. R. I). Schechtrr.an. L. .M.. Kim. C, M.. and .Kouri. R. II.
(19X1) Mulagcnic and carcinogenic potency of extracts of diesel
and related environmental emissions: Simultaneous transformation
• and mulagenesis in HALH/c 3T3 cells. £m'ir/w. Int. 5. 411. .
Ember. L. i'979) T\tt diex-l dilemma: HPA's difficult decision. Km-
rinv.^u.-i 21. 15-41.
Environmental Protection Agency (I97X) Health ei'fecls associated
with diesel exhaust emissions: Literature review and evaluation.
T.I'A 6tX)/l-7S-«K;3.."U. S. Umironricnlal Protection Agency, R'.':
search Triangle Park. NC. ... ..-.
Lewtas. j. <•/ al. t!98l).Mulagenic and carcinogenic potency o: ex-
tracts of diesel and related environmental emissions: Study design.
sample generation, colle.-tion. and preparation. Enrintn. Int. 5.
3S3.'
Mitchell. A. IX Simmon. V. r.. Morlelmans. K. E.. Riccio. E. S.. Jolz.
- M. M.. and Evans. E. L. (19X1) Mutagenic and carcinogenic.
potency of extracts of diesel and related environmental emissions:
In twu mutagcnesis aniMJNA damage. Knrmm. Inf. 5. *93.
Slaga. T. J.. Tripled', L. L. and Nesnow. S. (19X1) Mutagenic and
carcinogenic potency of extracts of diesel and related environmen-
tal emissions: Two stage carcinogcitesis in skin tu.^ior sensitive
mice (SENCAR)./:»M ir..n./n/. 5. 417.
Waters. M. D. Nesnow. S.. lluisingh. J. L.. Sandhu. S. S.. and
Claxt(tn, L. eds. (1979) Applti'aiit'n »/ Short- Term Huta.\\ays in lite
Frai'tttmuli'tn uihl Analysts of Cttniptex Knrirtaint.'nial Mi.\lurr\.
Plenum Press. NY.
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